My lab is interested in defining how the maternal load is established during oogenesis and decoded after fertilization. We know the identity of the most important maternal transcripts and maternally supplied RNA-binding proteins. We know that these factors are required for germline development, oocyte maturation, and pattern formation in early embryogenesis. But we do not yet know which proteins regulate which mRNAs, or what mechanisms coordinate this regulation in space and time. The wiring diagram is incomplete, and our mechanistic understanding limited. We have employed a protein-centric approach to mapping maternal RNA regulation in the nematode Caenorhabidits elegans. We have used a combination of 1) quantitative in vitro biochemistry, 2) in vitro evolution (SELEX), 3) high throughput RIP-seq experiments, and 4) functional assays in live invertebrate animals to define the sequence motifs recognized by a number of important maternal RNA-binding proteins. This work led to the identification of function cis-regulatory elements in some of the most important maternal mRNAs. The work made it clear that binding specificity is not sufficient to explain mRNA targeting in vivo. All proteins studied so far bind to short linear partially degenerate motifs present in 30-50% of all mRNAs. In some cases, the motifs have been shown to be necessary but not sufficient to drive regulatory activity. Putting a motif, even in multiple copies, into a transgene does not confer RBP-dependent regulation. Binding does not equal regulation; binding site context is also crucial for targeting. We have demonstrated that motifs for several RBPs cluster into a small sequence window in the 3UTR of a well studied maternal transcript encoding the C. elegans Notch homolog glp-1. This protein is required for germline progenitor cell proliferation in the germline, and anterior cell fate specification in the early embryo. The protein is expressed in a limited subset of germline and embryonic cells, buts its mRNA is found throughout the entire tissue and in all cells of the early embryo. We suspect that clustering provides the necessary context for regulation. Binding events that are cooperative can lead to higher stability and higher affinity interactions. Binding events that are competitive can be important for regulatory mechanisms that rely on recruiting cellular machinery, such as deadenylases, cytoplasmic polyA polymerases, and the translation initiation machinery. We have now shown that glp-1 mRNA is regulated through stable deadenylation in the early embryo. We have developed a number of key reagents, transgenic worm strains, mutants, and assays to investigate the role of repressive and activating RBPs in modulating the length of the polyA tail. We have also developed a robust and reliable strategy to make transgenic reporter animals in high throughput by library transgenesis. These methods enable functional characterization and mechanistic studies in vivo at a resolution that was previously impossible. Our innovative interdisciplinary approach, coupled to the strong atmosphere at UMass Medical School in RNA biology and C. elegans genetics, will ensure rapid progress in defining the maternal effect in embryogenesis at the functional level.